Tumor Escape Tumor Rejection Developing Tumor Anti-tumor Immune Response Suppression of the Anti-tumor Immune Response NKT Type I M1 Macrophages MDSCs M2 Macrophages NKT Type II MDSCs Tregs Tregs CD8 + T Cells NK Cells CD8 + T Cell Th1 Cell NK Cells Myeloid-derived Suppressor Cells
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Myeloid-derived Suppressor Cells...Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of immature myeloid progenitor cells that fail to differentiate into granulocytes,
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Tumor Escape
Tumor Rejection
Developing Tumor
Anti-tumor Immune Response
Suppression of the Anti-tumor Immune Response
NKT Type I
M1 Macrophages
MDSCs M2 Macrophages
NKT Type II
MDSCs
Tregs
Tregs
CD8+ T Cells
NK Cells
CD8+
T Cell
Th1 Cell
NK Cells
Myeloid-derived Suppressor Cells
T Cell
T Cells
T Cell
TCR-CD3 Complex
TCR-CD3 Complex
TCR-CD3 Complex
CD8
CCL2
CCL2
TIM-3
Inhibition of Th1 or CD8+ T Cell Responses
MacrophageRecruitment
Treg RecruitmentT Cell Arrest in G0 /G1
T Cell Apoptosis
Low L-arginine
T Cell
T CellA2A R
T CellPD-1
PD-L1
M2 Macrophages
NK Cells
CD8+ T Cells
Th17 Cell
iTreg CellnTreg Cells
IL-10TGF-βIL-35
IL-6IL-10TGF-β
IL-12 lowArg1
CCL22
TGF-βRetinoic Acid
IL-10TGF-βArg1
IL-10TGF-β
TGF-βROSArg1iNOS
NO
—Cys-NO
Peroxynitrite
Adenosine
Inhibition of T Cell Migration
↓ CD62L
ADAM17
Galectin-9 CD73AMP
MDSC
↑ Arg1NADPHOxidase
↑ iNOS
O2 NO
L-cystine
L-cystineLow L-cysteineLow L-arginine
L-arginine
Membrane-bound TGF-β
CATs
Inhibition of T Cell Growth and Differentiation
Production of Factors that Drive Regulatory T Cell Generation and
Immunosuppression
Nitrosylation of Target Proteins→ Inhibition of
IL-2 Signaling
Loss of TCR ζ Chain
Loss of TCR ζ Chain
Nitration or Nitrosylation of TCR, CD3, CD8, and CCL2
Inhibition of T Cell Proliferation, Migration, and Function
H2O2
Th1 or CD8+
T Cell
Inhibition of the Functions of NK Cells and
CD8+ T Cells
Myeloid-derived Suppressor CellsMyeloid-derived suppressor cells (MDSCs) are a heterogeneous population of immature myeloid progenitor cells that fail to differentiate into granulocytes, macrophages, and dendritic cells. These immature cells have the capacity to suppress immune responses mediated by natural killer cells, CD8+ and CD4+ T cells. MDSCs accumulate in the blood, bone marrow, and secondary lymphoid organs of tumor-bearing mice and cancer patients, where circulating levels of MDSCs have been shown to correlate with clinical stage, metastatic burden, and chemoresistance. As a result, these cells have been suggested to have a causative role in promoting tumor-associated immunosuppression. In mouse, MDSCs are broadly defined as CD11b+ Gr-1+ cells, but the relative expression levels of Ly-6G and Ly-6C define two specific subsets known as granulocytic and monocytic MDSCs. Mouse granulocytic MDSCs are CD11b+ Gr-1/Ly-6Ghigh Ly-6Clow, while monocytic MDSCs are CD11b+ Gr-1/Ly-6G–/low Ly-6Chigh. Human MDSCs also commonly express CD11b along with Siglec-3/CD33 and lack HLA-DR and the lineage markers CD3, CD14, CD19, and CD56. Similar to mouse, human granulocytic (Lin– CD11b+ CD14– CD15+ CD33+ CD66b+ HLA-DR–) and monocytic (Lin- CD11b+ CD14+ CD15– CD33+ CD66b– HLA-DR–) subsets have been identified, but they are based on the differential expression of CD14, CD15, and CD66b/CEACAM-8.
MDSCs are of great interest due to their immunosuppressive properties. While the mechanism by which MDSCs inhibit NK cells is currently not well-understood, multiple pathways are responsible for MDSC-mediated T cell suppression including production of arginase 1/ARG1 and upregulation of nitric oxide synthase 2 (iNOS). ARG1 and iNOS metabolize L-arginine and either together, or separately, cause the loss of the TCR zeta chain, promote nitration or nitrosylation of TCR, CD3, CD8, and CCL2, disrupt IL-2 signaling, and inhibit T cell proliferation. Additionally, MDSCs secrete immunosuppressive cytokines and induce regulatory T cell development. Although it is now evident that MDSCs are a major obstacle for immunotherapy, further characterization is necessary to determine how MDSCs accumulate, how they function, and mechanisms by which they can be inhibited. R&D Systems, Novus Biologicals, and Tocris Biosciences together offer a wide range of products for characterizing MDSCs and studying their functions.
MDSC–Mediated Mechanisms of Immunosuppression
Mouse Markers
Granulocytic MDSCs
Monocytic MDSCs
CD11b/Integrin aM + +
Gr-1/Ly-6G + -
Ly-6C - +
Human Markers
Granulocytic MDSCs
Monocytic MDSCs
Lin - -
CD11b/Integrin aM + +
CD33 + +
HLA-DR - -
Human Markers
Granulocytic MDSCs
Monocytic MDSCs
CD14 - +
CD15 + -
CD66b/CEACAM-8 + -
Analysis of Human Granulocytic and Monocytic MDSCs by Flow CytometryH
LA-D
R
Lineage cocktail
Rela
tive
cell
coun
t
Arginase 1/ARG1
Rela
tive
cell
coun
t
IDO
Inte
grin
αM
/CD
11b
Siglec-3/CD33Re
lativ
e ce
ll co
unt
Arginase 1/ARG1
Rela
tive
cell
coun
t
IDO
CD14
CEACAM-8/CD66b
Monocytic MDSCs
Granulocytic MDSCs
Identification of Human Granulocytic and Monocytic Myeloid-derived Suppressor Cells by Flow Cytometry. (A) Lin–/HLA-DR– cells were detected in human peripheral blood mononuclear cells by staining with a lineage cocktail containing Alexa Fluor 700-conjugated Mouse Anti-Human CD3e and CD19 Monoclonal Antibodies (R&D Systems, Catalog # FAB100N and # FAB4867N, respectively) and an Alexa Fluor 750-conjugated Mouse Anti-Human HLA-DR Monoclonal Antibody (R&D Systems, Catalog # FAB4869S). Lin–/HLA-DR– cells were gated. (B) CD11b+/CD33+ cells were detected in the Lin–/HLA-DR– cell population by staining with an APC-conjugated Mouse Anti-Human Siglec-3/CD33 Monoclonal Antibody (R&D Systems, Catalog # FAB1137A) and an Alexa Fluor 405-conjugated Mouse Anti-Human Integrin aM/CD11b Monoclonal Antibody (R&D Systems, Catalog # FAB16991V). CD11b+/CD33+ cells were gated. (C) Human granulocytic myeloid-derived suppressor cells (Lin–/CD11b+/CD14–/CD33+/CEACAM-8+/HLA-DR–) and monocytic myeloid-derived suppressor cells (Lin–/CD11b+/CD14+/CD33+/CEACAM-8–/HLA-DR–) were detected in the Lin–/HLA-DR–/CD11b+/CD33+ cell population by staining with a PE-conjugated Mouse Anti-Human CEACAM-8/CD66b Monoclonal Antibody (R&D Systems, Catalog # FAB4246P) and a PerCP-conjugated Mouse Anti-Human CD14 Monoclonal Antibody (R&D Systems, Catalog # FAB3832C).
Detection of IDO in Human Granulocytic and Monocytic Myeloid-derived Suppressor Cells by Flow Cytometry. Following identification of myeloid-derived suppressor cells (MDSCs) by flow cytometry, expression of Indoleamine 2,3-dioxygenase/IDO in the gated (A) granulocytic (Lin–/CD11b+/CD14–/CD33+/CEACAM-8+/HLA-DR–) and (B) monocytic (Lin–/CD11b+/CD14+/CD33+/CEACAM-8–/HLA-DR–) MDSC populations was determined by staining with an Alexa Fluor 488-conjugated Mouse Anti-Human Indoleamine 2,3-dioxygenase/IDO Monoclonal Antibody (R&D Systems, Catalog # IC6030G; filled histograms) or an Alexa Fluor 488-conjugated Mouse IgG1 Isotype Control (R&D Systems, Catalog # IC002G; open histograms).
Detection of Arginase 1 Expression in Human Granulocytic and Monocytic Myeloid-derived Suppressor Cells by Flow Cytometry. Following identification of myeloid-derived suppressor cells (MDSCs) by flow cytometry, expression of Arginase 1 in the gated (A) granulocytic (Lin–/CD11b+/CD14–/CD33+/CEACAM-8+/HLA-DR–) and (B) monocytic (Lin–/CD11b+/CD14+/CD33+/CEACAM-8–/HLA-DR–) MDSC populations was determined by staining with an Alexa Fluor 488-conjugated Mouse Anti-Human Arginase 1/ARG1 Monoclonal Antibody (R&D Systems, Catalog # IC8026G; filled histograms) or an Alexa Fluor 488-conjugated Mouse IgG2B Isotype Control (R&D Systems, Catalog # IC0041G; open histograms).
A
A
A
B
B
B
C
Analysis of Mouse Granulocytic and Monocytic MDSCs by Flow CytometryCD
11b
Gr-1
MonocyticGranulocytic
Identification of Mouse Monocytic and Granulocytic Myeloid-derived Suppressor Cells by Flow Cytometry. Mouse monocytic (CD11b+/Gr-1low/mid/Ly6C+) and granulocytic (CD11b+/Gr-1high/Ly6C–) myeloid-derived suppressor cells (MDSCs) from C57BL/6 mouse bone marrow cells were identified by staining with an APC-conjugated Rat Anti-Mouse Gr-1/Ly-6G Monoclonal Antibody (R&D Systems, Catalog # FAB1037A) and a PE-conjugated Rat Anti-Mouse Integrin aM/CD11b Monoclonal Antibody (R&D Systems, Catalog # FAB1124P). CD11b+/Gr-1low/mid and CD11b+/Gr-1high cells were gated to show the two populations of MDSCs.
Fluorochrome-conjugated & Unlabeled Antibodies from R&D Systems and Novus BiologicalsR&D Systems and Novus Biologicals offer a wide selection of unlabeled and fluorochrome-conjugated antibodies for the identification and characterization of mouse and human MDSCs.
Antibodies for Select Positive and Negative Markers used to Identify Granulocytic and Monocytic MDSCs by Flow Cytometry
Molecule Species Clone
Fluorochrome-conjugated Antibodies for Flow Cytometry (Catalog #s)
APC Fluores-cein PE PerCP
Alexa Fluor Additional Alexa Fluor conjugates
Unconjugated Antibodies
(Applications)488 700 350/405/594/647/750
CD3
Human UCHT1 FAB100A FAB100F FAB100P FAB100C FAB100G FAB100N FAB100V/FAB100T/FAB100R/FAB100S
▀ R&D Systems product ▀ Novus Biologicals productApplication Key: B/N Blocking/Neutralization ChiP Chromatim Immunoprecipitation Depl Depletion E ELISA FA Functional Assay FC Flow Cytometry ICC/IF Immunocytochemistry/Immunofluorescence IHC Immunohistochemistry IV In vitro IP Immunoprecipitation WB Western Blot
Antibodies Against Other Cell Surface & Intracellular Markers used for MDSC Identification or Characterization
Molecule Species Clone
Fluorochrome-conjugated Antibodies for Flow Cytometry (Catalog #s)
APC Fluorescein PE PerCPAlexa Fluor Additional Alexa Fluor
conjugatesUnconjugated
Antibodies (Applications)488 700 405/594/647/750
CD45 Human 2D1 FAB1430A FAB1430P FAB1430C FAB1430G FAB1430N MAB1430 (FC,
Fluorochrome-conjugated & Unlabeled Antibodies from R&D Systems and Novus Biologicals continued
▀ R&D Systems product ▀ Novus Biologicals productApplication Key: B/N Blocking/Neutralization ChiP Chromatim Immunoprecipitation Depl Depletion E ELISA FA Functional Assay FC Flow Cytometry ICC/IF Immunocytochemistry/Immunofluorescence IHC Immunohistochemistry IV In vitro IP Immunoprecipitation WB Western Blot
Recombinant Proteins & ELISA Kits from R&D SystemsR&D Systems portfolio includes recombinant proteins for generating MDSC-like cells from bone marrow-derived cells and ELISA Kits for detecting molecules that affect MDSC proliferation or promote immunosuppression.
Select Recombinant Proteins for Inducing MDSC-like Cells in vitro
Molecule Species Catalog #
G-CSFHuman 214-CS
Mouse 414-CS
GM-CSFHuman 215-GM
Mouse 415-ML
IL-4Human 204-IL
Mouse 404-ML
Select Recombinant Proteins for Inducing MDSC-like Cells in vitro
Molecule Species Catalog #
IL-6Human 206-IL
Mouse 406-ML
IL-13Human 213-ILB
Mouse 413-ML
* Recombinant Human G-CSF, GM-CSF, IL-4, and IL-6 are also available as Animal-Free™ proteins and GMP-grade proteins with the exception of G-CSF.
In addition to the single analyte ELISAs listed above, we also offer the membrane-based Proteome Profiler™ Human XL Cytokine Array (R&D Systems, Catalog # ARY022) and Mouse XL Cytokine Array (R&D Systems, Catalog # ARY028), as well as bead-based Luminex® Screening and Performance Assays, which can be used to simultaneously detect most or all of these analytes.
Select ELISAs for Detecting Molecules Secreted by MDSCs or Molecules in the Tumor Microenvironment that Affect MDSCs
Molecule Species Quantikine® ELISA Catalog #
DuoSet® ELISA Catalog #
G-CSFHuman DCS50 DY214
Mouse MCS00 DY414
GM-CSF Human DGM00 DY215
Mouse MGM00 DY415
IFN-gHuman DIF50 DY285
Mouse MIF00 DY485
IL-1bHuman DLB50 DY201
Mouse MLB00C DY401
IL-4Human D4050 DY204
Mouse M4000B DY404
IL-6Human D6050 DY206
Mouse M6000B DY406
Select ELISAs for Detecting Molecules Secreted by MDSCs or Molecules in the Tumor Microenvironment that Affect MDSCs
Molecule Species Quantikine® ELISA Catalog #
DuoSet® ELISA Catalog #
IL-10Human D1000B DY217B
Mouse M1000B DY417
IL-13Human D1300B DY213
Mouse M1300CB DY413
TGF-b1Human DB100B DY240
Mouse MB100B DY1679
TNF-aHuman DTA00C DY210
Mouse MTA00 DY410
VEGFHuman DVE00 DY293B
Mouse MMV00 DY4893
Antibodies Against Other Cell Surface & Intracellular Markers used for MDSC Identification or Characterization
Molecule Species Clone
Fluorochrome-conjugated Antibodies for Flow Cytometry (Catalog #s)
APC Fluorescein PE PerCPAlexa Fluor Additional Alexa Fluor
conjugatesUnconjugated
Antibodies (Applications)488 700 405/594/647/750
NCAM-1/CD56
Human 301040 FAB2408A FAB2408P MAB2408 (E, FC, WB)
Tocris Small Molecules for Myeloid-derived Suppressor Cell Research Tocris Biosciences offers a large selection of small molecules that can be used to study MDSC functions.
Tocris Small Molecules for Myeloid-Derived Suppressor Cell Research
T 0156 1676 Highly potent, selective PDE5 inhibitor
The reported activity on MDSCs is based on a publication by Najjar, Y.G. & J.H. Finke (2013) Front. Oncol. 3:49., except where indicated. ** Grabner B. et al. (2015) Nat. Commun. 6:6285. *** Talmadge J.E. & D.I. Gabrilovich (2013) Nat. Rev. Cancer 10:739.
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